Part:BBa_K1689011

dCas9-delta alpha

dCas9 fused with Δα segment of β-glactosidase

For their ease of use, low assay time, cost-effectiveness, high sensitivity, electrochemical biosensors have emerged as attractive methods for molecular detection. Combined with PC Reporter, we could further enhance it's applicability, that is, to implement pathogen diagnosis in settings outside clinics and without the need of specialized analytical laboratories.

β-glactosidase (LacZ) is an exoglycosidase from E.coli, composed of 1024 amino acids. LacZ is commonly used as reporter, known for blue white screen, which catalyzes X-gal to produce blue dye; by using different substrates, for example ONPG, reaction can be measured quantitatively.

In our project, we dissected LacZ as Δα, ∆ω segments to fuse with dCas9 to do the same as our PC Reporter.



β-galactosidase is used to catalyze the hydrolysis of p-aminophenyl-β-D-galactopyranoside (PAPG) to p-aminophenol (PAP), which can be oxidized at a working electrode held at 220mV versus the reference electrode.

References:
1 Mohler, W. A., & Blau, H. M. (1996). Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells. Proceedings of the National Academy of Sciences, 93(22), 12423-12427.
2 Biran, I., Klimentiy, L., Hengge-Aronis, R., Ron, E. Z., & Rishpon, J. (1999). On-line monitoring of gene expression. Microbiology, 145(8), 2129-2133.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1150
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 4
  Illegal BamHI site found at 3429
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]